Beneficial traits such as herbicide resistance, drought and stress tolerance, insect and pest resistance, phyto-remediation of soil contaminants, and horticultural qualities such as aluminum tolerance, stay-green appearance, pigmentation and growth habit are among a long list of features that can be improved in plants, including turfgrass, using transgene technology. However, the possibility of transgene escape from transgenic plants to wild and non-transformed species raises valid ecological concerns regarding commercialization of transgenic plants.
Although numerous risk assessment studies have been conducted on transgenic plants of aimual and/or self-pollinating crops (Ellstrand and Hoffman, 1990; Hoffman, 1990; Dale, 1992; 1993; Rogers and Parkes, 1995; Ellstrand et al., 1999; Altieri, 2000; Dale et al., 2002; Eastham and Sweet, 2002; Stewart et al., 2003; Pilson and Prendeville, 2004; Marvier and Van Acker, 2005), very little information is available on the potential risks from the commercialization and large-scale seed production of perennial transgenic grasses.
In a three-year field study on gene flow of transgenic bentgrass, it was observed that pollen from the transgenic nursery traveled at least 978 feet (Wipff and Friker, 2000; 2001). A recent landscape-level study on pollen-mediated gene flow from genetically modified creeping bentgrass demonstrated long-distance viable pollen movement from multiple source fields of genetically modified creeping bentgrass (Watrud et al., 2004). A subsequent study by the same group documented establishment and distribution of transgenic plants in wild populations (Reichman et al., 2006). Spatial distribution and parentage of transgenic plants (as confirmed by analyses of nuclear ITS and chloroplast matK gene trees) suggested that establishment had resulted from both pollen-mediated intraspecific hybridizations and from crop seed dispersal. These results demonstrate that transgene flow from short-term production can result in establishment of transgenic plants at multi-kilometer distances from genetically modified source fields or plants (Reichman et al., 2006). Therefore, there is a need to develop methods that decrease, or even prevent transgene escape in a production field of transgenic plants before large-scale commercialization of such transgenic plants, including turfgrass.
In flowering plants, gene flow can occur through movement of pollen grains and seeds, with pollen flow often contributing the major component. With the availability of current molecular technologies, various gene containment strategies have been developed to alter gene flow by interfering with flower pollination, fertilization, and/or fruit development (Daniell, 2002). If transgenic plants can also be engineered as male sterile, there will be no viable pollen grains produced from the transgenic plants, thus preventing the potential risk of transgene escape into the surrounding environment by out-crossing with non-transgenic plants or wild species.
Site-specific recombination is a process involving reciprocal exchange between specific nucleic acid sites (referred to as target sites) catalyzed by specialized proteins known as site-specific recombinases (Craig, 1988). These recombinases can alter genomic DNA sequences in specific ways, providing powerful tools for the development of a new generation of molecular technology for crop improvement. Site-specific recombinases recognize specific DNA sequences, and in the presence of specific recombination sites they catalyze the recombination of DNA strands (Ow and Medberry, 1995). In these site-specific recombination systems, recombinases can catalyze excision or inversion of a DNA fragment according to the orientation of their specific target sites. Recombination between directly oriented sites leads to excision of the DNA between them, whereas recombination between inverted target sites causes inversion of the DNA between them
The lambda integrase family of site-specific recombination systems consists of more than 100 different members. Among the most prominent of these are lambda Int, Cre/lox, FLP/FRT, R/RS, and Gin/gix. Recombinases such as Cre, FLP, R and Gin catalyze DNA recombination between their respective DNA substrates or target sites, loxP, FRT, RS and gix. These recombination systems use a common reaction pathway to carry out very different biological functions. They utilize a single polypeptide recombinase capable of recognizing a small DNA sequence without requiring any accessory factors. Cre/lox, FLP/FRT, R/RS, Gin/gix and λ-Int are probably the most utilized systems for genetic manipulation of plants and animals. In heterologous systems, Cre/lox, FLP/FRT, R/RS, Gin/gix carry out a freely reversible reaction, whereas λ-Int requires additional factors to carry out the reverse reaction. The minimal length of a loxP and FRT site is 34 bp (Hoess et al., 1982; Jayaram, 1985), and both of these consist of two 13-bp inverted repeats surrounding an 8-bp spacer region (boxed, see below), which confers directionality.

In plants, the application of two site-specific recombination systems, the FLP/FRT system from the 2 μm plasmid of the eukaryote yeast (Broach et al., 1982) and the Cre/lox system from prokaryotic bacteriophage P1 (Austin et al., 1981), have been studied most extensively (Odell and Russell, 1994; Ow and Medberry, 1995; Luo and Kausch, 2002). The primary natural function of the Cre/lox and FLP/FRT systems is related to the amplification of extrachromosomal DNA molecules (bacteriophage or plasmid) in bacteria and yeast cells, respectively (Austin et al., 1981; Sadowski, 1995). Unlike another member of the integrase family of the site-specific recombinases, bacteriophage λ integrase (Argos et al., 1986), Cre and FLP recombinases do not require additional factors for controlling site-specific recombination reactions (Cox, 1983; Huang et al., 1991), making them good candidates for applications in heterologous organisms. The basic features of the FLP/FRT and the Cre/lox systems are that recombinases can catalyze the inversion, excision, and/or integration of nucleic acid fragments.
The Cre/lox recombination system from bacteriophage P1 was the first system evaluated in plant cells for its functionality in site-specific DNA recombination. In 1990, Dale and Ow demonstrated that Cre recombinase could excise, invert, or integrate extrachromosomal DNA molecules in tobacco protoplasts. In the same year, Odell et al. (1990) provided other crucial evidence that the Cre gene could be stably expressed in plant cells (tobacco), and that the Cre protein could recognize and recombine lox sites integrated into the plant genomic DNA. Further, the Cre gene has been shown to be successfully passed from one plant to another through cross-pollination
The FLP/FRT recombination system, which functions endogenously in eukaryotic yeast cells, was also identified as having the capability of catalyzing efficient recombination reactions in heterologous eukaryotic cells (Golic and Lindquist, 1989; O'Gorman et al., 1991). Lyznik et al. (1993) used a modified FLP coding sequence from pOG44 (O'Gorman et al., 1991) to synthesize a chimeric plant FLP gene driven by the maize ubiquitin promoter to show activity of FLP recombinase in maize and rice cells. In 1994, Lloyd and Davis published a report on FLP-mediated activation of a hygromycin resistance gene in the tobacco genome by cross-pollination. Soon after, Lyznik et al. (1995; 1996) demonstrated that the activity of FLP/FRT system could be controlled in a precise manner in maize cells with high molecular fidelity.
Thus, both FLP/FRT and Cre/lox site-specific recombination systems have been shown, for example, to function not only in bacteria, yeast, insect cells, mammalian cells (Cox, 1983; Golic and Lindquist, 1989; Huang et al., 1991; O'Gonnan et al., 1991; Chou and Perrimon, 1992; Rong and Golic, 2000), but also in tobacco (Lloyd and Davis, 1994; Qin et a1.1994; Bar et al., 1996), Arabidopsis (Odell et al., 1990; Dale and Ow, 1991; Bayley et al., 1992; Russel et al., 1992; Kilby et al., 1995; Osborne et al., 1995; Sonti et al., 1995; Luo et al., 2000), turfgrass (Luo et al., 2002; Hu et al., 2006), tomato (Stuurman et al., 1996; Zhang et al., 2006), maize and rice (Lyznik et al., 1993; 1995; 1996; Srivastava and Ow, 2001; Hoa et al., 2002; Toriyama et al., 2003; Sreekala et al., 2005), potato (Cuellar et al., 2006) and wheat (Srivastava et al., 1999).
Accordingly, in order to advance transgenic technologies for plant genetic improvement without the undesirable effects of gene flow, it would be useful to have a system in which a transgenic plant can be produced which has reduced or no sexual reproductive capability and further, in which unwanted transgenic nucleotide sequences are no longer present in the transgenic plant. Thus, the present invention provides methods and compositions wherein a dual recombination system is employed to achieve controlled excision of unwanted transgenic DNA and self excision of recombinase-coding genes in plants, thereby improving applications of plant transgenic technologies.